Revised Abstract: The INK4a/ARF locus encodes two tumor suppressors, p165/INK4a and p19ARF (p14ARF in humans) that regulate the functions of the retinoblastoma protein (Rb) and the p53 transcription factor, respectively. p16/INK4A is an inhibitor of the cyclin D-specific kinases, cdk4 and cdk6, whose phosphorylation of Rb in the G1 phase of the cell division cycle helps to cancer its growth suppressive functions, thereby enabling cells to enter S phase. p19ARF binds to the p53 negative regulator Mdm2, inhibiting its E3 ubiquitin ligase activity and sequestering it in the nucleolus. This enables p53 to accumulate in the nucleoplasm where is activates the transcription of genes, including Mdm2 and p21/CIP1, that induce either cell cycle arrest or apoptosis depending on the biologic context. Rb, p53 and INK4a/ARF are the most frequently disrupted genes in human cancer, regardless of patient age or tumor type. The goal of this proposal is to further understand how INK4a/ARF mediates in tumor suppressive functions by studying its regulation, downstream targets and genetic modifiers in different physiologic contexts in mice. Although four INK4 genes encoding related cdk4/6 inhibitors have been identified, only p16/INK4a has been strongly implicated as a potent tumor suppressor in humans. However, mice lacking INK4a/ARF or ARF alone exhibit a similar cancer prone phenotype, suggesting that p16/INK4a may play a subtler role in tumor suppression in this species. I propose to address this question by creating INK4a-null mice that retain ARF function. In turn, I hope to address this question by creating INK4a- null mice that retain ARF function. In turn, I hope to create conditional ARF knock-out strains that more closely model the role of this gene in particular human cancers. Although the p19/ARF-Mdm2-p53 pathway is frequently disrupted in tumors, both p19ARF and Mdm2, are each likely to have other targets. Preliminary data suggest that the ARF-Mdm2 interaction is bi-directional with each protein affecting the activity of the other. I hope to explore this feature in greater detail, taking advantage of recently cloned Mdm2 isoforms, naturally expressed in Emu-Myc mouse lymphomas, which do not bind to p53. Moreover, although cells lacking p53 alone are resistant to ARF's effects, we have now found that enforced expression of p19ARF in cells lacking both Mdm2 and p53 (with our without ARF) induces cell cycle arrest. I offer a number of strategies aimed to identify novel ARF targets, other than Mdm2. In summary, I propose to focus on the following unresolved questions: Is p16 INK4a a tumor suppressor in mice and in what cell types does it act? What genetic alterations cooperate with ARF loss in tumorigenesis? Does Mdm2 over-expression synergize with ARF-loss in promoting tumors, and how do unusual isoforms of Mdm2 contribute? With what targets, apart from Mdm2, does p19ARF interact to induce cell cycle arrest? These studies address many poorly understood features of INK4a-ARF signaling that should have direct relevance to many forms of human cancer.